Recombinant Photobacterium profundum tRNA pseudouridine synthase B (truB)

What is Photobacterium profundum and why is it significant for truB research?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae. It is particularly notable as a model organism for studying adaptations to extreme environments. P. profundum strain SS9, originally isolated from the Sulu Sea in 1986, is characterized by optimal growth at 15°C and 28 MPa (approximately 280 atmospheres), making it both a psychrophile and piezophile (pressure-loving organism) .

The bacterium has rod-shaped cells measuring 2-4μm in length and 0.8-1.0μm in width, with a single unsheathed flagellum. Its genome consists of two circular chromosomes, similar to other members of the Photobacterium genus . The ability of P. profundum to thrive under high hydrostatic pressure makes its molecular machinery, including truB, particularly interesting for understanding adaptations to extreme conditions.

Beyond strain SS9, there are three other cultured wild-type strains: 3TCK (isolated from San Diego Bay, with optimal growth at 9°C and 0.1 MPa), DSJ4 (from the Ryukyu Trench at 5110m depth, with optimal growth at 10°C and 10 MPa), and strain 1230 . This diversity of growth optima allows for comparative studies of pressure adaptation mechanisms.

What is the function of tRNA pseudouridine synthase B (truB) in bacterial systems?

TruB is an enzyme that catalyzes the site-specific isomerization of uridine to pseudouridine at position 55 in the TΨC loop of tRNA molecules. This modification is one of the most evolutionarily conserved RNA modifications found across all domains of life .

The TruB enzyme from P. profundum specifically functions by:

• Recognizing the TΨC stem-loop structure in tRNA

• Catalyzing the rotation of the uracil base at position 55

• Breaking the N1-glycosidic bond

• Reforming a C5-glycosidic bond to create pseudouridine

This isomerization does not alter the base-pairing properties of the nucleoside but enhances base stacking and stabilizes RNA secondary structure due to the additional hydrogen bonding capability of pseudouridine. While the deletion of truB in some organisms like E. coli does not prevent growth under standard conditions, it does reduce competitive fitness and impairs survival during temperature shifts, suggesting its importance for adaptation to environmental stresses .

How does pseudouridine modification affect tRNA structure and function?

Pseudouridine modification at position 55 in tRNA molecules significantly impacts both structural stability and functional properties through several mechanisms:

• Enhanced Base Stacking: The C5-glycosidic bond in pseudouridine allows for improved base stacking interactions, which strengthens the tertiary structure of tRNA.

• Additional Hydrogen Bonding: Pseudouridine contains an additional imino group (NH) that can form hydrogen bonds with water molecules, contributing to structural rigidity.

• Stabilization of RNA Structure: Studies have shown that pseudouridine modifications increase the thermal stability of RNA helices and the melting temperature of tRNA, which may be particularly advantageous for organisms living under extreme conditions.

• Conformational Rigidity: The presence of pseudouridine in the TΨC loop restricts conformational flexibility, potentially protecting tRNA from degradation under stress conditions.

Research with E. coli has demonstrated that while truB deletion mutants grow normally under standard conditions, they show competitive disadvantages during co-culture with wild-type strains and exhibit defects in surviving rapid temperature transitions (from 37°C to 50°C) . This suggests that the pseudouridine modification is particularly important during environmental stress, making it a potentially crucial adaptation for pressure-tolerant organisms like P. profundum.

How can researchers assess the enzymatic activity of recombinant truB in vitro?

Researchers can assess the enzymatic activity of recombinant P. profundum truB using several complementary approaches:

1. Radioactive Assay:

• Synthesize [32P]UTP-labeled tRNA substrate via in vitro transcription

• Incubate labeled substrate with purified truB enzyme

• Extract and digest RNA with nuclease P1

• Analyze resulting nucleotides via two-dimensional thin-layer chromatography (2D-TLC)

• Compare against known pseudouridine standards

• Quantify pseudouridine formation by autoradiography

2. CMCT-Primer Extension Assay:

• Treat RNA with N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT)

• This reagent forms adducts with pseudouridine residues

• Perform reverse transcription with a labeled primer

• The CMCT-Ψ adduct causes reverse transcriptase to stop one nucleotide before the modified position

• Analyze extension products on sequencing gels

• Compare band patterns between truB-treated and control samples

3. HPLC Analysis:

• Prepare tRNA substrate via in vitro transcription

• Incubate with purified truB enzyme

• Digest treated RNA to nucleosides

• Analyze by high-performance liquid chromatography

• Compare retention times with pseudouridine standards

• Quantify peaks to determine pseudouridine content

4. Nearest Neighbor Analysis:

• Synthesize RNA with one nucleotide radioactively labeled

• Treat with truB enzyme

• Digest with RNase T2 (which produces 3'-monophosphates)

• Analyze products by 2D-TLC

• Identify modified nucleotides by their position relative to the labeled nucleotide

For optimal activity assessment, researchers should consider:

• Including appropriate positive and negative controls

• Testing activity under different buffer conditions (pH 7.0-8.5)

• Evaluating temperature dependence (10-37°C)

• Assessing pressure effects on enzymatic activity (0.1-30 MPa)

What experimental setup is required for studying truB function under high-pressure conditions?

To effectively study truB function under high pressure, researchers should implement specialized equipment and protocols:

High-Pressure Experimental Setup:

• Hardware Requirements:

  • High-pressure vessels capable of maintaining stable pressures up to 70 MPa

  • Temperature control system (typically 0-25°C range for P. profundum studies)

  • Pressure generation system (hydraulic pump or gas booster)

  • Pressure monitoring devices with digital recording capabilities

  • Sample containment system (typically polyethylene transfer pipettes or polyethylene tubing sealed with no headspace)

• Experimental Approaches:

  • In vitro enzymatic assays: Conduct standard truB activity assays in pressure-resistant containers

  • Growth studies: Compare growth of wild-type and truB mutant strains under various pressure conditions

  • Gene expression analysis: Extract RNA from cells grown under different pressures to assess truB expression levels

  • Protein stability studies: Analyze thermal and pressure denaturation profiles of purified truB

• Protocol for High-Pressure Growth Experiments:

  • Prepare cultures in appropriate media (e.g., 2216 medium for marine bacteria)

  • Fill pressure-resistant containers completely to eliminate air bubbles

  • Seal containers without trapped air

  • Place in pressure vessel and pressurize to desired level (e.g., 0.1, 10, 28, 70 MPa)

  • Incubate at appropriate temperature (typically 15°C for SS9 strain)

  • Monitor growth by sacrificing parallel samples at designated timepoints

• Gene Complementation Approach:

  • For assessing truB function, construct deletion mutants using gene disruption methods

  • Test growth under pressure (e.g., 280 atm/28 MPa for SS9)

  • Complement mutants with plasmid-borne truB gene

  • Assess restoration of high-pressure growth phenotype

Based on studies with pressure-sensitive genes, it's important to note that different experimental setups may yield variable results. For instance, in P. profundum studies, when exconjugants were tested for high-pressure growth ability after gene complementation, they were typically incubated at 15°C for 3-5 days before results could be assessed .

How does P. profundum truB structure and function compare to homologs from non-piezophilic organisms?

P. profundum truB presents several distinctive features compared to its homologs from non-piezophilic organisms, reflecting adaptations to high-pressure environments:

Structural Comparisons:

• Amino Acid Substitutions: Specific substitutions that favor protein stability under pressure, such as reduced void volumes and increased ion pairs

• Modified Flexibility: Strategic distribution of rigid and flexible regions to accommodate pressure effects

• Hydration Properties: Altered surface properties that modulate protein-water interactions under pressure

What is the relationship between truB activity and high-pressure growth phenotypes in P. profundum?

While the search results don't directly establish a relationship between truB activity and high-pressure growth in P. profundum specifically, evidence from related studies suggests important connections:

Key Observations:

• In Alcanivorax dieselolei, a marine oil-degrading bacterium, truB expression was significantly upregulated (log2 fold change of 3.09) under mild hydrostatic pressure (10 MPa) compared to atmospheric pressure, making it one of the most enhanced genes in the entire transcriptome .

• This upregulation suggests that tRNA modification by truB may be crucial for protein synthesis under pressure conditions.

• Protein synthesis is known to be a pressure-sensitive process, with one of the most vulnerable steps being the binding of aminoacyl-tRNA to ribosomes, as it requires a conformational change leading to volume increase (an unfavorable process under pressure) .

• The formation of pseudouridine at position 55 in tRNA likely plays a role in stabilizing tRNA structure under pressure conditions, potentially facilitating more efficient translation.

Hypothesized Mechanism:

Under high-pressure conditions, cellular processes involving volume increases are inhibited. Translation, particularly the binding of aminoacyl-tRNA to ribosomes, is sensitive to pressure because it involves conformational changes with positive volume changes. The pseudouridine modifications introduced by truB stabilize tRNA structure, potentially counteracting some pressure effects and maintaining translation efficiency.

In P. profundum specifically, truB activity may be part of a suite of adaptations that allow efficient protein synthesis under high pressure. While RecD function has been directly linked to high-pressure growth in P. profundum , the specific contribution of truB remains to be fully characterized through targeted gene deletion and complementation studies similar to those performed for RecD.

How can researchers construct and validate truB mutants in P. profundum?

Based on established methods for generating gene deletions in P. profundum and related bacteria, the following approach is recommended for constructing and validating truB mutants:

Construction of truB Gene Disruption Mutants:

• PCR Amplification of Target Sequence:

  • Design primers to amplify an internal portion of the truB gene

  • Typical PCR conditions: 92°C for 1 min, 48°C for 1 min, and 72°C for 1 min for 25 cycles

• Cloning into Suicide Vector:

  • Clone PCR product into an intermediate vector (e.g., pCR2.1)

  • Subclone into a suicide vector (e.g., pMUT100) that cannot replicate in P. profundum

  • The suicide vector should contain a selectable marker (e.g., kanamycin resistance)

• Conjugal Transfer:

  • Transform constructed plasmid into donor E. coli strain (e.g., MC1061 containing helper plasmids)

  • Perform conjugation with P. profundum:

    • Harvest cells by centrifugation

    • Resuspend in appropriate medium

    • Spot onto membrane filters on agar plates

    • Incubate at room temperature for 12-16 hours

    • Wash cells from filters and plate onto selective medium

    • Incubate at 15°C for 3-5 days

• Selection of Integration Mutants:

  • Select exconjugants on media containing appropriate antibiotic

  • Since the plasmid cannot replicate in P. profundum, antibiotic resistance indicates integration into the genome via homologous recombination

  • This results in disruption of the target gene

Validation of truB Mutants:

• PCR Verification:

  • Design primers that anneal outside and within the deleted/disrupted region

  • Confirm gene disruption by PCR analysis of genomic DNA

• Functional Validation:

  • Analyze tRNA modifications using HPLC or 2D-TLC to confirm absence of pseudouridine at position 55

  • Perform CMCT-primer extension assays to detect presence/absence of pseudouridine in specific tRNA positions

• Phenotypic Analysis:

  • Test growth under varying pressure conditions (0.1-70 MPa)

  • Compare growth rates and cell morphology of mutant versus wild-type strains

  • Assess competitive fitness in co-culture experiments

• Complementation Tests:

  • Clone intact truB gene into broad-host-range plasmid

  • Introduce into truB mutant strain via conjugation

  • Verify restoration of pseudouridine formation

  • Test whether high-pressure growth defects are rescued

This comprehensive approach will allow researchers to establish the specific role of truB in P. profundum's adaptation to high-pressure environments.

What techniques can be used to study the effects of pressure on truB enzyme kinetics?

Studying enzyme kinetics under pressure requires specialized equipment and experimental approaches. The following methodologies are recommended for investigating truB activity under varying pressure conditions:

High-Pressure Enzyme Kinetics Methods:

• Stopped-Flow Spectroscopy Under Pressure:

  • Utilize high-pressure stopped-flow apparatus with sapphire windows

  • Monitor reaction progress using fluorescence detection if possible

  • Determine initial reaction rates at different substrate concentrations

  • Calculate kinetic parameters (Km, kcat) at various pressures

  • Plot activation volume (ΔV‡) based on pressure effects on rate constants

• Real-Time NMR Under Pressure:

  • Use specialized high-pressure NMR cells

  • Monitor reaction progress directly through chemical shift changes

  • Determine rate constants at various pressures

  • Particularly useful for studying the isomerization reaction catalyzed by truB

• High-Pressure Quench-Flow System:

  • React enzyme and substrate under pressure

  • Rapidly quench at defined time points

  • Analyze reaction products after depressurization

  • Use HPLC or mass spectrometry for product quantification

  • Determine reaction rates at different pressures

• Comparative Analysis Approach:

  • Perform parallel experiments with truB from P. profundum and non-piezophilic organisms

  • Compare pressure dependence of kinetic parameters

  • Identify differences that may relate to pressure adaptation

Data Analysis and Interpretation:

The pressure dependence of reaction rate constants (k) can be analyzed using the transition state theory equation:

$\left(\frac{\partial \ln k}{\partial P}\right)_T = -\frac{\Delta V^‡}{RT}$

Where:

• ΔV‡ is the activation volume

• R is the gas constant

• T is absolute temperature

• P is pressure

Negative activation volumes indicate that pressure accelerates the reaction, while positive values indicate inhibition by pressure. For truB from piezophilic organisms like P. profundum, you might expect smaller positive (or even negative) activation volumes compared to enzymes from non-piezophilic sources, reflecting adaptation to high-pressure environments.

How can insights from P. profundum truB research advance understanding of RNA modification systems?

Research on P. profundum truB offers unique insights that extend beyond this specific enzyme to broader understanding of RNA modification systems:

• Evolutionary Adaptations in RNA Modification Enzymes:

  • P. profundum truB represents an adaptation of a conserved RNA modification system to extreme conditions

  • Comparative analysis with homologs from organisms living at different pressures can reveal how RNA modification enzymes evolve under selective pressure

  • This provides a natural experiment for understanding the evolutionary plasticity of RNA modification systems

• Functional Importance of RNA Modifications Under Stress:

  • While truB deletion in E. coli only affects growth under stress conditions, its potential essentiality for high-pressure growth in piezophiles would highlight the critical nature of RNA modifications under extreme conditions

  • This may reveal previously unappreciated roles of specific RNA modifications in cellular stress responses

• Structural Stability Mechanisms in RNA:

  • Understanding how pseudouridine modifications contribute to tRNA stability under pressure offers insights into fundamental principles of RNA stabilization

  • These principles may apply to other structured RNAs and have implications for RNA-based therapeutics and technologies

• Translation Systems Engineering:

  • Insights from piezophilic truB could inform the design of translation systems functioning under non-standard conditions

  • Applications may include cold-adapted or pressure-resistant protein expression systems for biotechnology

What methodological approaches can assess the impact of truB modifications on global translation under pressure?

To comprehensively assess how truB-mediated tRNA modifications affect translation under pressure, researchers should employ multi-omics approaches:

1. Ribosome Profiling Under Pressure:

• Culture cells (wild-type and truB mutants) under various pressure conditions

• Treat with antibiotics that freeze ribosomes on mRNA

• Isolate ribosome-protected fragments

• Sequence to determine ribosome positions genome-wide

• Analyze translation efficiency and pausing at specific codons

• Compare translation profiles between wild-type and truB mutant strains

2. Proteomics Analysis:

• Perform quantitative proteomics on wild-type and truB mutant strains grown under different pressure conditions

• Identify differentially expressed proteins

• Look for patterns related to specific codon usage or mRNA structural features

• Correlate with transcriptomics data to distinguish translational from transcriptional effects

3. tRNA Modification Analysis:

• Isolate total tRNA from cells grown under different pressure conditions

• Analyze the complete set of tRNA modifications using mass spectrometry

• Compare modification profiles between atmospheric and high-pressure conditions

• Quantify changes in pseudouridylation at position 55 and potential compensatory modifications

4. In vitro Translation Systems:

• Develop cell-free translation systems using components from P. profundum

• Compare translation efficiency using native tRNAs versus tRNAs lacking pseudouridine modifications

• Perform experiments under varying pressure conditions in specialized high-pressure vessels

• Measure translation rates and accuracy under different conditions

5. Codon-Specific Translation Efficiency:

• Design reporter constructs with different codon usage patterns

• Express in wild-type and truB mutant strains under various pressure conditions

• Quantify protein output and correlate with codon usage

• Identify whether specific codons are more affected by the absence of truB activity

These approaches would provide a comprehensive understanding of how truB-mediated tRNA modifications contribute to translation efficiency under pressure, potentially revealing codon-specific effects and mechanisms of translational adaptation to high-pressure environments.

How might structural studies of P. profundum truB inform the engineering of pressure-adapted enzymes?

Structural studies of P. profundum truB could provide valuable insights for the rational design of pressure-adapted enzymes for biotechnological applications:

Key Structural Features with Engineering Implications:

• Cavity Architecture:

  • High-pressure adapted proteins often have reduced internal cavities

  • Mapping the void volumes in P. profundum truB and comparing with non-piezophilic homologs

  • This information could guide protein engineering approaches for filling cavities in pressure-sensitive enzymes

• Flexibility and Rigidity Distribution:

  • Pressure-adapted enzymes often have altered distribution of rigid and flexible regions

  • Structural analysis of B-factors and molecular dynamics simulations

  • Identifying flexible regions that allow conformational adaptation without compromising catalytic function

• Surface Hydration Properties:

  • Pressure affects protein-water interactions and hydration layers

  • Analyzing surface charge distribution and hydrophobic/hydrophilic patches

  • Engineering surface properties to optimize function under pressure

• Salt Bridges and Electrostatic Interactions:

  • Pressure-adapted proteins often have increased numbers of ion pairs

  • Identifying unique salt bridge networks in P. profundum truB

  • Implementing similar stabilizing interactions in other enzymes

Engineering Applications:

• Biocatalysis Under Pressure:

  • High-pressure processes can offer advantages in industrial biocatalysis

  • Engineered pressure-resistant enzymes could expand the operating conditions for biocatalytic processes

  • Principles from P. profundum truB could guide development of pressure-stable biocatalysts

• Cold-Adapted Enzymes:

  • Many principles of pressure adaptation overlap with cold adaptation

  • Structural insights could inform the development of enzymes for low-temperature applications

  • Combined pressure and cold adaptation for specific industrial processes

• Protein Therapeutics Stability:

  • Principles of structural stability under pressure could improve storage stability of protein therapeutics

  • Engineering longer shelf-life and stress resistance into protein drugs

• Structure-Based Design Strategy:

  • Develop a systematic approach for pressure-adapting enzymes based on truB structural features

  • Create a pressure-adaptation "toolkit" of structural modifications

  • Apply to industrially relevant enzymes to improve their performance under non-standard conditions

Recent cryo-EM studies of other proteins from P. profundum, such as the SiaQM transporter , have demonstrated the value of structural approaches for understanding piezophilic adaptations. Similar high-resolution structural studies of truB would significantly advance the field of enzyme engineering for extreme conditions.

How does truB function compare with other tRNA modification enzymes in P. profundum?

While the available search results don't provide comprehensive information about all tRNA modification enzymes in P. profundum specifically, we can make evidence-based comparisons based on known patterns in related organisms:

Comparative Analysis of tRNA Modification Systems:

In P. profundum, truB likely plays a particularly important role compared to other tRNA modification enzymes because:

• The pseudouridine at position 55 is one of the most universally conserved RNA modifications, suggesting fundamental importance

• Studies in E. coli have shown that combining truB mutations with mutations affecting TrmA (which modifies the adjacent position 54) significantly increases temperature sensitivity , suggesting functional interaction between these modifications

• In A. dieselolei, truB was among the most upregulated genes under pressure conditions , indicating potential specialization for high-pressure adaptation

• Other piezophilic bacteria may have evolved different strategies for stabilizing tRNA, potentially involving different modification patterns or enzyme activities

The specific constellation of tRNA modifications in P. profundum likely represents an adapted system that maintains translation efficiency under high-pressure conditions. Detailed comparative analysis of the complete set of tRNA modifications in P. profundum grown under different pressure conditions would provide further insights into this specialized system.

What is known about truB interaction with other RNA modification pathways under pressure conditions?

Potential Interaction Mechanisms:

• Modification Hierarchy:

  • In many RNA modification pathways, the presence of one modification can affect the efficiency of subsequent modifications

  • The pseudouridine at position 55 introduced by truB might create structural conditions necessary for other modifications

  • Under pressure conditions, this hierarchy might be particularly important for maintaining tRNA function

• Compensatory Modifications:

  • In the absence of truB activity, other modification enzymes might increase activity to compensate

  • For example, adjacent modifications like m⁵U54 (introduced by TrmA) might be upregulated

  • These compensatory mechanisms could be pressure-dependent

• Co-regulation of Modification Enzymes:

  • RNA modification enzymes are often co-regulated under stress conditions

  • Pressure stress might trigger coordinated changes in multiple modification pathways

  • The significant upregulation of truB observed in A. dieselolei under pressure might be part of a broader response

Research Approaches to Study Interactions:

• Multi-enzyme Deletion Studies:

  • Generate single and combined deletions of truB and other RNA modification enzymes

  • Test growth and translation efficiency under various pressure conditions

  • Look for synthetic phenotypes that reveal functional interactions

• RNA Modification Profiling:

  • Use mass spectrometry to profile all tRNA modifications in wild-type and truB mutant strains

  • Compare modification patterns under atmospheric versus high-pressure conditions

  • Identify modifications that change in response to truB deletion

• Protein-Protein Interaction Studies:

  • Use co-immunoprecipitation or bacterial two-hybrid systems to identify proteins that interact with truB

  • Determine whether these interactions are pressure-dependent

  • Look for potential modification enzyme complexes

• Transcriptional Network Analysis:

  • Examine co-expression patterns of RNA modification enzymes under pressure

  • Identify potential shared regulatory mechanisms

  • Map the RNA modification stress response network

The study of interaction networks among RNA modification enzymes under pressure represents an important frontier in understanding piezophilic adaptation mechanisms, with truB likely playing a central role in this network.

What are the most promising future research directions for P. profundum truB studies?

Based on current knowledge gaps and potential applications, several high-priority research directions emerge for P. profundum truB:

• Structure-Function Relationships Under Pressure:

  • Determine high-resolution structures of P. profundum truB under varying pressure conditions

  • Identify pressure-sensing regions within the enzyme

  • Elucidate the molecular mechanisms of maintained catalytic activity under pressure

• Systematic Mutagenesis Studies:

  • Create point mutations in P. profundum truB to identify residues critical for pressure adaptation

  • Engineer pressure-sensitivity into the enzyme to create pressure-sensing variants

  • Test chimeric enzymes with domains from piezophilic and non-piezophilic homologs

• Transcriptome-Wide Impact of truB:

  • Analyze ribosome profiling data from wild-type and truB mutant strains under pressure

  • Identify specific mRNAs whose translation is most affected by loss of truB activity

  • Determine whether pressure adaptation involves specialized translation of specific stress-response genes

• Evolutionary Studies:

  • Compare truB sequences across marine bacteria from different depths

  • Identify convergent adaptations to high pressure in different bacterial lineages

  • Reconstruct the evolutionary history of pressure adaptation in tRNA modification systems

• Systems Biology Approach:

  • Map the complete network of RNA modifications and their interactions under pressure

  • Model how these modification networks respond to changing environmental conditions

  • Develop predictive frameworks for understanding adaptation to extreme environments

These research directions would not only advance our understanding of P. profundum truB specifically but would also contribute to broader knowledge about RNA biology, extremophile adaptations, and the fundamental principles of protein function under non-standard conditions.

How might understanding P. profundum truB contribute to deep-sea biotechnology applications?

The study of P. profundum truB offers several promising avenues for biotechnological applications related to deep-sea environments:

• Pressure-Resistant Enzyme Technologies:

  • Principles derived from truB structure could inform the design of pressure-stable enzymes

  • Applications in deep-sea resource utilization and bioremediation

  • Development of biocatalysts for high-pressure industrial processes

• Expression Systems for Deep-Sea Environments:

  • Creation of pressure-adapted protein expression systems incorporating truB and related components

  • In situ protein production for deep-sea applications

  • Biotechnological tools for studying deep-sea ecosystems

• RNA-Based Technologies:

  • Development of pressure-stable RNA-based therapeutics or diagnostic tools

  • RNA storage and delivery systems incorporating stability principles from tRNA modifications

  • RNA catalysts (ribozymes) with enhanced stability under extreme conditions

• Biomimetic Materials:

  • Design of pressure-resistant biomaterials inspired by molecular adaptations in piezophiles

  • Self-assembling systems that respond to pressure changes

  • Nanomaterials with pressure-dependent properties for sensing applications

• Bioremediation Applications:

  • Engineered microorganisms with enhanced performance under deep-sea conditions

  • Oil-spill remediation technologies incorporating pressure-adapted enzymes

  • Degradation of pollutants at high pressure and low temperature